Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure ... [more ▼]

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure. Broad spectrum inhibitors of these enzymes such as SAHA can inhibit tumor growth both in vitro and in vivo and are currently used as anti-cancer agents in clinic. For many years, we are investigating the specific role of individual HDAC members in cancer biology and we have recently demonstrated that depletion of HDAC5 using siRNA technology triggered cancer cells to both autophagy and apoptosis (ref papier). The study of autophagy in cancer is a new research field that has recently generated tremendous attention due to the recognition that autophagy can have either pro-survival or pro-death functions depending on its level of activation. In addition, more and more studies indicate that a complex relationship exists between autophagy and apoptosis, and that the interplay between these two processes determines whether a cell will live or die. Aims: The goal of this study is to further understand the role of autophagy induced by HDAC5 depletion. Current investigations include determining the molecular mechanisms by which HDAC5 depletion induces autophagy and exploring regulatory relationship between autophagy and apoptosis on cancer cell death in absence of HDAC5. Results: The set up of the autophagy in absence of HDAC5 was demonstrated by the conversion of LC3 and development of autophagosomes by electronic microscopy. Transcriptomic study demonstrated a deregulation of a set of genes involved in ROS detoxification in HDAC5 depleted cancer cells leading to significant increase of ROS levels. Further investigations showed that pretreatment with NAC, a ROS scavenger, effectively blocked the accumulation of ROS and autopahgy triggered by HDAC5 silencing. Moreover, HDAC5 depletion induces activation of JNK, and knockdown of JNK by siRNA inhibited ROS production and autophagy, but antioxidant NAC failed to block JNK activation induced by HDAC5 depletion indicating that JNK activation may be a upstream signaling of ROS and should be a core component in HDAC5 silencing-induced autophagic signaling pathway. Finally, blocking of autophagy induced by HDAC5 silencing with NAC or chloroquine and bafilomycin enhanced pro-apoptotic effect. Conclusion: Autophagy functions as a prosurvival mechanism to mitigate HDAC5 depletion-induced apoptotic cell death, suggesting that targeting autophagy might improve the therapeutic effects of specific HDAC5 inhibition. [less ▲]

in Congress of the International Society of Thrombosis and Hemostasis- 57th Annual SSC Meeting (2011, July)

Atherosclerosis is the principal cause of mortality in industrialized countries. Its development is influenced by several mediators of which thromboxane A(2) (TXA(2)) and 8-iso-PGF(2() have recently ... [more ▼]

Atherosclerosis is the principal cause of mortality in industrialized countries. Its development is influenced by several mediators of which thromboxane A(2) (TXA(2)) and 8-iso-PGF(2() have recently received a lot of attention. This study aimed to investigate the effect of a dual thromboxane synthase inhibitor and thromboxane receptor antagonist (BM-573) and ASA on lesion formation in apolipoprotein E-deficient mice. The combination of ASA and BM-573 was also studied. Plasma measurements demonstrated that the treatments did not affect body weight or plasma cholesterol levels. BM-573, but not ASA, significantly decreased atherogenic lesions as demonstrated by macroscopic analysis. Both treatments alone inhibited TXB(2) synthesis but only BM-573 and the combination therapy were able to decrease firstly, plasma levels of soluble intracellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) and secondly, the expression of these proteins in the aortic root of Apo E. These results were confirmed in endothelial cell cultures derived from human saphenous vein endothelial cells (HSVECs). In these cells, BM-573 also prevented the increased mRNA expression of ICAM-1 and VCAM-1 induced by U-46619 and 8-iso-PGF(2(). Our results show that a molecule combining receptor antagonism and thromboxane synthase inhibition is more efficient in delaying atherosclerosis in Apo E(-/-) mice than sole inhibition of TXA(2) formation. [less ▲]

Atherosclerosis is the principal cause of mortality in industrialized countries. Its development is influenced by several mediators of which thromboxane A(2) (TXA(2)) and 8-iso-PGF(2() have recently ... [more ▼]

Atherosclerosis is the principal cause of mortality in industrialized countries. Its development is influenced by several mediators of which thromboxane A(2) (TXA(2)) and 8-iso-PGF(2() have recently received a lot of attention. This study aimed to investigate the effect of a dual thromboxane synthase inhibitor and thromboxane receptor antagonist (BM-573) and ASA on lesion formation in apolipoprotein E-deficient mice. The combination of ASA and BM-573 was also studied. Plasma measurements demonstrated that the treatments did not affect body weight or plasma cholesterol levels. BM-573, but not ASA, significantly decreased atherogenic lesions as demonstrated by macroscopic analysis. Both treatments alone inhibited TXB(2) synthesis but only BM-573 and the combination therapy were able to decrease firstly, plasma levels of soluble intracellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) and secondly, the expression of these proteins in the aortic root of Apo E. These results were confirmed in endothelial cell cultures derived from human saphenous vein endothelial cells (HSVECs). In these cells, BM-573 also prevented the increased mRNA expression of ICAM-1 and VCAM-1 induced by U-46619 and 8-iso-PGF(2(). Our results show that a molecule combining receptor antagonism and thromboxane synthase inhibition is more efficient in delaying atherosclerosis in Apo E(-/-) mice than sole inhibition of TXA(2) formation. [less ▲]

Tumor hypoxia is a common characteristic of most solid tumors and is correlated with poor prognosis for patients partly because hypoxia promotes resistance to cancer therapy. Hypoxia selects cancer cells ... [more ▼]

Tumor hypoxia is a common characteristic of most solid tumors and is correlated with poor prognosis for patients partly because hypoxia promotes resistance to cancer therapy. Hypoxia selects cancer cells that are resistant to apoptosis and allows the onset of mechanisms that promote cancer cells survival including autophagy. Previously, we showed that human hepatoma HepG2 cells were protected under hypoxia against the etoposide-induced apoptosis. In this study, respective putative contribution of autophagy and BNIP3 in the protection conferred by hypoxia against the etoposide-induced apoptosis was investigated. We report that autophagy is induced by etoposide, a process that is not affected by hypoxic conditions. Using Atg5 siRNA, we show that etoposide-induced autophagy promotes apoptotic cell death under normoxia but not under hypoxia. Then, we investigated whether the hypoxia-induced protein BNIP3 could explain the different effect of autophagy on cell death under hypoxia or normoxia. We show that the silencing of BNIP3 does not affect autophagy whatever the pO2 but participates in the protective effect of hypoxia against etoposide-induced apoptosis. Together, these results suggest that autophagy might be involved in etoposide-induced cell death only under normoxia and that BNIP3 is a major effector of the protective mechanism conferred by hypoxia to protect cancer cells against etoposide-induced apoptotic cell death. [less ▲]

in Neoplasia : An International Journal for Oncology Research (2009), 11

Tumor hypoxia is one of the features of tumor microenvironment that contributes to chemoresistance in particular by cellular adaptations that modulate the apoptotic process. However, the mechanisms ... [more ▼]

Tumor hypoxia is one of the features of tumor microenvironment that contributes to chemoresistance in particular by cellular adaptations that modulate the apoptotic process. However, the mechanisms involved in this resistance still need deeper understanding. In this study, we investigated the involvement of four transcription factors, c-Myc, nuclear factor κB (NF-κB), p53, and c-jun/activator protein 1 (AP-1) in the hypoxia-induced resistance to etoposide in HepG2 cells. Whereas the profile of c-Myc and NF-κB activity did not fit the effect of hypoxia on caspase 3 activity, hypoxia decreased basal p53 abundance and DNA binding activity as well as p53 etoposide-induced activation. Short interfering RNA (siRNA) silencing evidenced that p53 was required for etoposide-induced apoptosis under normoxia. An inhibition of its activity under hypoxia could thus be responsible at least in part for the protection observed under hypoxic conditions. Moreover, p53 was found to induce the expression of Bak1. We showed that Bak1 was involved in the etoposide-induced apoptosis because Bak1 siRNA decreased it. Conversely, hypoxia increased c-jun DNA binding activity in the presence of etoposide. siRNA-mediated silencing of c-jun increased the responsiveness of cells to etoposide under hypoxia, as shown by an increase in caspase 3 activity and lactate dehydrogenase release. These effects occurred in a p53-independent manner. These data evidenced that hypoxia decreased the responsiveness of HepG2 cells to etoposide at least by two independent pathways involving p53 inhibition and c-jun activation. [less ▲]

A solid tumour forms an organ-like structure that is comprised of cancer cells as well as stroma cells (fibroblasts, inflammatory cells) that are embedded in an extracellular matrix and are nourished by ... [more ▼]

A solid tumour forms an organ-like structure that is comprised of cancer cells as well as stroma cells (fibroblasts, inflammatory cells) that are embedded in an extracellular matrix and are nourished by vascular network. However, tumoral microenvironment is heterogeneous due to the abnormal vasculature network and high proliferation rate of cancer cells. Because of these features, some regions are starved from oxygen, a phenomenon called hypoxia. Transient hypoxia is associated with inadequate blood flow while chronic hypoxia is the consequence of the increased oxygen diffusion distance due to tumour expansion. Both types of hypoxia are correlated with poor outcome for patients. Moreover, hypoxia also enhances chemoresistance of cancer cells. Firstly, the delivery of drugs in hypoxic area and cellular uptake of it are affected by hypoxia or associated acidity. Secondly, some chemotherapeutic drugs require oxygen to generate free radicals that contribute to cytotoxicity. Last, hypoxia induces cellular adaptations that compromise the effectiveness of chemotherapy. In response to nutrient deprivation due to hypoxia, the rate of proliferation of cancer cells decreases but chemotherapeutic drugs are more effective against proliferating cells. On the other hand, hypoxia induces adaptation by post-translational and transcriptional changes that promote cell survival and resistance to chemotherapy. Through these changes, hypoxia promotes angiogenesis, shift to glycolytic metabolism, expression of ABC transporters, cell survival by inducing the expression of genes encoding growth factors and the modulation of apoptotic process. The aim of this review is to provide a description of known hypoxia-induced mechanisms of chemoresistance at a cellular level. [less ▲]

Background: it is now well established that hypoxia renders tumor cells resistant to radio- but also chemotherapy. However, few elements are currently available as for the mechanisms underlying this ... [more ▼]

Background: it is now well established that hypoxia renders tumor cells resistant to radio- but also chemotherapy. However, few elements are currently available as for the mechanisms underlying this protection. Results: in this study, physiological hypoxia was shown to inhibit apoptosis induced in HepG2 cells by etoposide. Indeed, hypoxia reduced DNA fragmentation, caspase activation and PARP cleavage. The DNA binding activity of 10 transcription factors was followed while the actual transcriptional activity was measured using specific reporter plasmids. Of note is the inhibition of the etoposideinduced activation of p53 under hypoxia. In parallel, data from low density DNA microarrays indicate that the expression of several pro- and anti-apoptotic genes was modified, among which are Bax and Bak whose expression profile paralleled p53 activity. Cluster analysis of data unravels several possible pathways involved in the hypoxia-induced protection against etoposide-induced apoptosis: one of them could be the inhibition of p53 activity under hypoxia since caspase 3 activity parallels Bax and Bak expression profile. Moreover, specific downregulation of HIF-1α by RNA interference significantly enhanced apoptosis under hypoxia possibly by preventing the hypoxia mediated decrease in Bak expression without altering Bax expression. Conclusion: these results are a clear demonstration that hypoxia has a direct protective effect on apoptotic cell death. Moreover, molecular profiling points to putative pathways responsible for tumor growth in challenging environmental conditions and cancer cell resistance to chemotherapeutic agents. [less ▲]

Background: It is more and more recognized that hypoxia plays a role in the resistance of cancer cells to chemotherapy. However, the mechanisms underlying this resistance still need deeper understanding ... [more ▼]

Background: It is more and more recognized that hypoxia plays a role in the resistance of cancer cells to chemotherapy. However, the mechanisms underlying this resistance still need deeper understanding. The aim of this study was to investigate the effect of hypoxia on this process since hypoxia is one of the hallmarks of tumor environment. Results: The effect of hypoxia on the apoptosis induced by etoposide, one drug commonly used in chemotherapy, was investigated using three different cancer cell lines. Gene expression changes were also studied in order to delineate the mechanisms responsible for the hypoxia-induced chemoresistance. We observed that hypoxia differentially influenced etoposide-induced cell death according to the cancer cell type. While hypoxia inhibited apoptosis in hepatoma HepG2 cells, it had no influence in lung carcinoma A549 cells and further enhanced it in breast cancer MCF-7 cells. Etoposide increased p53 activity in all cell lines while hypoxia alone decreased it only in HepG2 cells. Hypoxia had no influence on the etoposide-induced p53 activity in A549, increased p53 abundance in MCF-7 cells but markedly decreased p53 activity in HepG2 cells. Using low density DNA arrays to detect the expression of genes involved in the regulation of apoptosis, etoposide and hypoxia were shown to each influence the expression of numerous genes, many of the ones influenced by etoposide being p53 target genes. Again, the influence of hypoxia on the etoposideinduced changes was different according to the cell type. Conclusion: These results evidenced that there was a striking parallelism between the effect of hypoxia on the etoposide-induced p53 stabilization as well as p53 target gene expression and its effect on the etoposide-induced apoptosis according to the cell type. They are very interesting not only because they provide one possible mechanism for the induction of chemoresistance under hypoxic conditions in cells like HepG2 but also because they indicate that not all cell types respond the same way. This knowledge is of importance in designing adequate treatment according to the type of tumors. [less ▲]

Hypoxia is a common feature in tumors associated with an increased resistance of tumor cells to therapies. In addition to O2 diffusion–limited hypoxia, another form of tumor hypoxia characterized by ... [more ▼]

Hypoxia is a common feature in tumors associated with an increased resistance of tumor cells to therapies. In addition to O2 diffusion–limited hypoxia, another form of tumor hypoxia characterized by fluctuating changes in pO2 within the disorganized tumor vascular network is described. Here, we postulated that this form of intermittent hypoxia promotes endothelial cell survival, thereby extending the concept of hypoxia-driven resistance to the tumor vasculature. We found that endothelial cell exposure to cycles of hypoxia reoxygenation not only rendered them resistant to proapoptotic stresses, including serum deprivation and radiotherapy, but also increased their capacity to migrate and organize in tubes. By contrast, prolonged hypoxia failed to exert protective effects and even seemed deleterious when combined with radiotherapy. The use of hypoxia-inducible factor-1α (HIF-1α)–targeting small interfering RNA led us to document that the accumulation of HIF-1α during intermittent hypoxia accounted for the higher resistance of endothelial cells. We also used an in vivo approach to enforce intermittent hypoxia in tumor-bearing mice and found that it was associated with less radiation-induced apoptosis within both the vascular and the tumor cell compartments (versus normoxia or prolonged hypoxia). Radioresistance was further ascertained by an increased rate of tumor regrowth in irradiated mice preexposed to intermittent hypoxia and confirmed in vitro using distinctly radiosensitive tumor cell lines. In conclusion, we have documented that intermittent hypoxia may condition endothelial cells and tumor cells in such a way that they are more resistant to apoptosis and more prone to participate in tumor progression. Our observations also underscore the potential of drugs targeting HIF-1α to resensitize the tumor vasculature to anticancer treatments. (Cancer Res 2006; 66(24): 11736-44) [less ▲]

Tumor hypoxia has been described to increase the resistance of cancer cells to radiation therapy and chemotherapy. It also supports the invasiveness and metastatic potential of the tumor. However, few ... [more ▼]

Tumor hypoxia has been described to increase the resistance of cancer cells to radiation therapy and chemotherapy. It also supports the invasiveness and metastatic potential of the tumor. However, few data are available on the transduction pathway set up under hypoxia and leading to this resistance against anti-cancer therapies. HIF-1, the main transcription factor activated by hypoxia, has been recently shown to participate to this process although its role as an anti- or a pro-apoptotic protein is still controversy. In this study, we showed that hypoxia protected HepG2 cells against etoposide-induced apoptosis. The effect of hypoxia on cell death was assayed by measuring different parameters of the apoptotic pathway, like DNA fragmentation, caspase activity and PARP-1 cleavage. The possible implication of HIF-1 in the anti-apoptotic role of hypoxia was investigated using HIF-1α siRNA. Our results indicated that HIF-1 is not involved in the hypoxia-induced antiapoptotic pathway. Another transcription factor, AP-1, was studied for its potential role in the hypoxia-induced protection against apoptosis. Specific inhibition of AP-1 decreased the protection effect of hypoxia against etoposide-induced apoptosis. Together, all these data underline that hypoxia could mediate its anti-apoptotic role via different transcription factors depending on the cellular context and pro-apoptotic stimuli. [less ▲]

in Apoptosis : An International Journal on Programmed Cell Death (2006), 11

Apoptosis has become recognized as a crucial mechanism involved in a wide range of physiological and pathological processes. Following an initial pro-apoptotic signal, controlling phases allow the cell to ... [more ▼]

Apoptosis has become recognized as a crucial mechanism involved in a wide range of physiological and pathological processes. Following an initial pro-apoptotic signal, controlling phases allow the cell to reinforce or downgrade signals leading to the irrevocable entry into apoptosis. Bak (Bcl-2-antagonist killer) is a mitochondrial pore-forming pro-apoptotic effector inhibited through titration by the antiapoptotic protein Mcl-1 (Myeloid cell leukemia-1). Viruses have taken advantage of proteasome-dependent degradation of Bak as a mechanism to prevent apoptosis in infected cells. It is not clear however whether regulation of Bak protein level is involved in other physiological processes. In this report, we show that Mcl-1 level is paralleled by Bak while a Mcl-1 non-interacting mutant of Bak does not accumulate in cells. This mechanism is proteasome independent. Following serum withdrawal, Bak accumulation becomes independent of Mcl-1 level and cells are sensitized to proapoptotic stimuli. Based on these results, we propose that regulation of Mcl-1-Bak steochiometry is a control mechanism used as a checkpoint to prevent or allow entry into apoptosis. [less ▲]

Increased levels of Mcl-1 (myeloid cell factor-1) have been reported in several cancers, suggesting an important role played by Mcl-1 in cancer cell survival. Mcl-1 is an anti-apoptotic protein shown to ... [more ▼]

Increased levels of Mcl-1 (myeloid cell factor-1) have been reported in several cancers, suggesting an important role played by Mcl-1 in cancer cell survival. Mcl-1 is an anti-apoptotic protein shown to delay or block apoptosis. In this work, using semiquantitative immunofluorescence, real-time PCR, and RNase protection assay, an increase in Mcl-1 expression was detected in hepatoma HepG2 cells incubated under hypoxia or in the presence of cobalt chloride. Through analysis of the Mcl-1 promoter sequence, a putative HIF-1 (hypoxiainducible factor-1) binding site was identified. A Mcl-1 promoter fragment containing this hypoxia-responsive element was able to bind HIF-1 in vitro. It also induced hypoxia-dependent transcription of a luciferase reporter gene, which was suppressed by anti-HIF-1 short interfering RNA. Finally, overexpression of Mcl-1 protected HepG2 cells against apoptosis induced by tertbutyl hydroperoxide as shown by inhibition of caspase-3 activation and DNA fragmentation. All these data suggest a potential anti-apoptotic role of HIF-1 that could protect cells against apoptosis under hypoxia by overexpression of the Mcl-1 protein. [less ▲]

in International Journal of Cancer = Journal International du Cancer (2005), 117(5), 764-74

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that plays a major role in cellular adaptation to hypoxia. The mechanisms regulating HIF-1 activity occurs at multiple levels in ... [more ▼]

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that plays a major role in cellular adaptation to hypoxia. The mechanisms regulating HIF-1 activity occurs at multiple levels in vivo. The HIF-1alpha subunit is highly sensible to oxygen and is rapidly degraded by the proteasome 26S in normoxia. Activation in hypoxia occurs through a multistep process including inhibition of HIF-1alpha degradation, but also increase in the transactivation activity of HIF-1. Several data indicate that phosphorylation could play a role in this regulation. In this report, we investigated the role of casein kinase 2 (CK2), an ubiquitous serine/threonine kinase, in the regulation of HIF-1 activity. Hypoxia was capable of increasing the expression of the beta subunit of CK2, of inducing a relocalization of this subunit at the plasma membrane, of inducing nuclear translocation of the alpha subunit and of increasing CK2 activity. Three inhibitors of this kinase, DRB (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole), TBB (4,5,6,7-tetrabromotriazole) and apigenin, as well as overexpression of a partial dominant negative mutant of CK2alpha, were shown to inhibit HIF-1 activity as measured by a reporter assay and through hypoxia-induced VEGF and aldolase expression. This does not occur at the stabilization process since they did not affect HIF-1alpha protein level. DNA-binding activity was also not inhibited. We conclude that CK2 is an important regulator of HIF-1 transcriptional activity but the mechanism of this regulation remains to be determined. Since HIF-1 plays a major role in tumor angiogenesis and since CK2 has been described to be overexpressed in tumor cells, this new pathway of regulation can be one more way for tumor cells to survive. [less ▲]